Retinal vascular diseases are major causes of vision loss in the United States and around the world. Age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity are all associated with the growth of new blood vessels (neovascularization) into or on the surface of the retina. They are also associated with the leakage of fluid from the retina blood vessels into the retina (edema) and with bleeding from the new vessels. To better treat these disorders, we need to understand the signaling pathways that control the growth and integrity of retinal blood vessels. We recently discovered a new signaling system that controls the growth of retinal blood vessels. In this pathway, a protein, Norrin, is secreted by Muller glial cells, the most abundant type of glial cell in the retina. Norrin binds with high affinity to a receptor protein, Frizzled4, which, together with two other proteins, Lrp5 and Tspan12, are present on the surface membrane of vascular cells. In humans and in mice, mutations in any of the genes encoding these four proteins lead to insufficient retinal vascular development. The objectives of this proposal are to characterize the response of retinal vascular cells to Norrin/Frizzled4 signaling. We will determine the response to Norrin/Frizzled4 signaling when it is initiated or terminated at different times during development, as well as the role of Norrin/Frizzled4 signaling in neovascularization. Within vascular cells, Norrin/Frizzled4 signaling induces a distinct set of changes in gene expression. We propose to characterize the network of transcription factors that control this response, beginning with one transcription factor, Sox17, which appears to play a central role. We will also explore the role of Norrin/Frizzled4 signaling in the interactions between vascular endothelial cells (the cells that line the inner face of the blood vessels) and pericytes (cells that unsheathe and stabilize the endothelial cells), and between endothelial cells and astrocytes, which form a scaffold along which the endothelial cells migrate as the retinal blood vessels develop. As the retinal vasculature is very similar between mice and humans, and the Norrin/Frizzled4 pathway (as well as other signaling pathways) is highly conserved, our experiments with mice should translate to humans. The experimental methods used include: studying genetically engineered mice in which genes can be activated or inactivated at different times, generating vascular lesions with a laser to study neovasculization, identifying which genes are direct targets of transcription factor binding, and studying the behavior of purified retinal vascular endothelial cells, pericytes, and astrocytes grown outside of the living animal. 1